Date of download: 6/30/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica Arch Neurol. 2010;67(10): doi: /archneurol Bioassay to determine antibodies to human native aquaporin 4 (AQP4). A, Strategy to establish the bioassay for the detection of antibodies binding to native AQP4. The glioma cell line LN18 was stably transduced with either the M1 isoform of the AQP4 gene (to establish the LN18 AQP4 cell line) or an empty vector (to establish the LN18 CTR control cell line) using a lentiviral expression system (the LN18 AQP4-M23 cell line expressing the M23 variant of AQP4 was produced likewise). The different antibodies used in the experiments are displayed. bp indicates base pairs. Expression of AQP4 was confirmed by Western blot of LN18 AQP4 and LN18 CTR cell lysates (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (B), immunocytochemistry (C), and flow cytometry on permeabilized cells (primary antibody: polyclonal rabbit anti-AQP4 antibody; secondary antibody: goat antirabbit IgG) (D). Figure Legend:

Date of download: 6/30/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica Arch Neurol. 2010;67(10): doi: /archneurol Detection of serum autoantibodies specific for native aquaporin 4. A, Immunofluorescence staining (original magnification ×10 [upper row] and ×63 [lower row]) was performed on LN18 CTR (left) and LN18 AQP4 (right) cells with serum from a neuromyelitis optica (NMO)–IgG–positive patient as the primary antibody source and Alexa Fluor-488–labeled goat antihuman IgG (Invitrogen Corp, Carlsbad, California) as the secondary antibody. B, Flow cytometry was performed with serum from a patient with multiple sclerosis (NMO-IgG negative [upper histogram]) and a patient with NMO (NMO-IgG positive [lower histogram]) on LN18 AQP4 (green) and LN18 CTR (red) cells. The difference in the median fluorescence intensity (MFI) obtained with the LN18 AQP4 and LN18 CTR cell lines is termed ΔMFI and corresponds to the concentration of aquaporin 4–specific serum antibodies. Sera from NMO-IgG–positive patients were tested at different concentrations for native aquaporin 4–specific antibodies in the bioassay. A representative example (C) and the dilution curves of 4 sera (D) are shown. Figure Legend:

Date of download: 6/30/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica Arch Neurol. 2010;67(10): doi: /archneurol Prevalence and isotype analysis of autoantibodies to native aquaporin 4 (nAQP4). A, Sera of different patients stratified according to their clinical diagnosis were analyzed for the prevalence of nAQP4-IgG. Each diamond represents 1 sample. The difference in median fluorescence intensity (ΔMFI) is shown for each group and the diagnostic cutoff is indicated by the dashed line. OND indicates other neurological disease; MS, multiple sclerosis; NMO, neuromyelitis optica; LETM, longitudinally extensive transverse myelitis; and RION, relapsing inflammatory optic neuritis. B, In 7 paired cerebrospinal fluid (CSF) and serum samples, anti-nAQP4- IgG antibodies were determined in parallel after adjusting for total IgG concentration. Corresponding CSF and serum ΔMFIs are plotted in relation to antibody indices (AIs) of 1.0 and 1.5. C, Analysis of IgG isotypes and IgM antibodies to nAQP4 is shown in 4 representative patients. D, Paired analysis of anti-nAQP4-IgG1 isotype levels are shown in relation to anti-nAQP4-IgG in positive samples. Figure Legend:

Date of download: 6/30/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Quantification and Functional Characterization of Antibodies to Native Aquaporin 4 in Neuromyelitis Optica Arch Neurol. 2010;67(10): doi: /archneurol Cytotoxic activity of native aquaporin 4 (nAQP4)–IgG. A, The LN18 AQP4 and LN18 CTR cells were incubated with different concentrations of total IgG purified from a high-titer anti-nAQP4-IgG serum (125 μg/mL, 62.5 μg/mL, 25 μg/mL, and 1.25 μg/mL). Survival of LN18 cells in the presence of natural killer (NK) cells was determined after 12 hours. B, The LN18 AQP4 and LN18 CTR cells were incubated with 125-μg/mL total IgG purified from anti-nAQP4-IgG–positive sera of patients with neuromyelitis optica or anti- nAQP4-IgG–negative patients with multiple sclerosis (MS). Survival of LN18 cells in the presence of NK cells was determined after 12 hours and normalized to the negative control sample (LN18 cells alone). The experiment was performed in duplicate; mean and standard deviation are shown. C, Correlation of the antibody-dependent cell-mediated cytotoxic in vitro activity (cell killing of LN18 AQP4 minus cell killing of LN18 CTR in the presence of serum IgG and NK cells) with different anti-nAQP4-IgG concentrations. Each dot represents the analysis of 1 patient's serum. D, The LN18 AQP4 and LN18 CTR cells were incubated with 125-μg/mL total IgG purified from nAQP4-IgG–positive sera of patients with NMO or nAQP4-IgG–negative sera of patients. Survival of LN18 cells in the presence of anti-nAQP4-IgG–negative serum as a complement source was determined after 12 hours and normalized to the negative control sample (LN18 cells alone). Figure Legend: